Biology

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This is the collection for the University of Waterloo's Department of Biology.

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“AnnoTools”: Extending AnnoTree and AnnoView for Database-Wide Genome Annotation, Visualization, and Comparison
(University of Waterloo, 2025-01-24) Tan, Huagang; Doxey, Andrew
Genomic analysis has revolutionized our understanding of the biology and evolutionary history of bacterial and archaeal microorganisms, leading to numerous applications in biotechnology, medicine, and environmental sciences. One of the fundamental aspects of genomic analysis is protein functional annotation, which involves assigning biological functions to protein-coding sequences identified within genomes. These annotations are widely used to support analyses, such as examining gene or function distributions across the tree of life and comparing gene neighborhoods across taxa. By combining these analyses, researchers can comprehensively explore gene functions and the mechanisms of given genes or gene clusters. In this thesis, I will introduce a pipeline that supports genomic analysis. The project consists of three parts: data annotation, visualization, and the language model. The first part of the pipeline is the generation of protein function annotations. Raw protein sequence data is downloaded from the Genome Taxonomy Database (GTDB) and submitted to two tools: Kofamscan and DIAMOND. Kofamscan assigns KEGG ORTHOLOGY IDs to each input sequence, while DIAMOND assigns Uniref IDs, which are then mapped to InterPro IDs. Combining these IDs provides comprehensive and reliable annotations. The data is filtered for quality and stored on a remote server as an annotation database for further analysis. The second part of the pipeline involves updating two user-friendly, web-based visualization tools, AnnoTree and AnnoView, which utilize the annotation database. AnnoTree displays the distribution and taxonomy of different protein annotations across GTDB using a tree of life representation, offering insights into biological and evolutionary patterns through species phylogenies and supporting genome-wide co-occurrence analysis. AnnoView focuses on comparing and exploring gene neighborhoods, identifying functionally related genes clustered together in genomes as "gene clusters," thus emphasizing window-based co-occurrence analysis. The new annotation database not only provides more comprehensive and accurate annotations, enhancing the databases that both visualization tools rely on, but also extends their functionalities for fast data retrieval and new features. The last part of the pipeline involves the application of the Word2Vec language model, which treats genome contigs as sentences in natural language and trains the model using the annotation database. After training, the updated model can encode each annotation from a specific protein family into high-dimensional vectors with continuous number, allowing researchers to explore annotations that share similar genomic contexts. This allows protein functions prediction based on this comparative gene neighborhood analysis. Finally, I will use one protein domain in the Type VI Secretion System (T6SS) as a case study. T6SS is a cell envelope-spanning machine that translocates toxic effector proteins into eukaryotic and prokaryotic cells. Besides the conserved essential core components, there are various effector and accessory proteins in the system. Some proteins are annotated as Domains of Unknown Function (DUF) and are poorly explored. In this case, I will focus on PF20598 (DUF6795), which shares a similar genomic context with one of the T6SS proteins. Using the visualization tools AnnoTree and AnnoView, I will demonstrate that this DUF is part of the T6SS cluster, supporting the hypothesis that it may function as an adaptor protein in T6SS. In summary, the AnnoTools pipeline integrates all components to enhance comparative genomic analysis with a large-scale annotation database. The user-friendly web-based tools enable researchers to visualize data both genome-wide and at a window-based scale. The ultimate goal of this thesis is to provide researchers with a comprehensive and easy-to-use method for predicting functions of genes or gene clusters of interest.
Determination of the accumulation of chiral pharmaceuticals (venlafaxine and 𝘖-desmethylvenlafaxine) in rainbow darters (𝘌𝘵𝘩𝘦𝘰𝘴𝘵𝘰𝘮𝘢 𝘤𝘢𝘦𝘳𝘶𝘭𝘦𝘶𝘮)
(University of Waterloo, 2025-01-22) Kowalczyk, Sarah; Servos, Mark
Pharmaceuticals are widespread contaminants of concern that enter the aquatic environment mainly via wastewater effluent. Over 50% of common pharmaceuticals are chiral, and this is notable due to the potential impact of chirality on the distribution, fate, and toxicity of these compounds. Historically, chirality has been overlooked when completing environmental risk assessments. The chiral antidepressant venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (desVEN) are pseudo-persistent through wastewater treatment and are not removed completely, being detected in surface waters globally at levels greater than 2.0 µg/L. At these concentrations, VEN and desVEN have been shown to impact the behavior, metabolism, population structures, and other biological responses of aquatic biota in receiving environments. The possible enantioselective bioaccumulation of each enantiomer of VEN (R and S) and desVEN (R and S) needs further investigation, as they are normally treated as racemic mixtures. An optimized and unbiased extraction method for individual enantiomers (R and S) of VEN and desVEN was developed. The extraction method and all subsequent sample cleanup was evaluated for the recoveries of VEN and desVEN enantiomers and possible enantiomeric bias. Accelerated solvent extraction (ASE) using acidified acetonitrile (1% formic acid by volume) was determined to be an acceptable extraction method with regards to the recoveries and chromatography for all VEN and desVEN enantiomers (performing better in comparison to ultrasonic solvent extraction, USE). The selection and mass of a fat retainer included in extraction cells for on-line extract cleanup and subsequent sample cleanup protocols were also optimized to reduce matrix effects (e.g., ion suppression) associated with co-eluates during liquid chromatography tandem mass spectrometry (LC-MS/MS). The addition of 2.5 g of neutral aluminum oxide during ASE had no enantioselective effects on extraction but resulted in better recoveries for all enantiomers of VEN and desVEN. Subsequent extract cleanup via solid-phase extraction (SPE) using Hydrophilic-Lipophilic-Balanced (HLB) cartridges had the highest recoveries in comparison to other SPE cartridges, liquid-liquid extraction, and QuEChERS, and no enantioselective effects were observed after analysis. Fish tissue mass up to 2.4 g and a final extract volume of 0.5 mL were chosen as the best compromise for a method with satisfactory sensitivity and minimal enantiomeric bias during extraction, while avoiding detrimental ion suppression for all VEN and desVEN enantiomers (with good absolute recovery, and extraction efficiency). The method detection limits (MDLs) for the final method ranged from 0.03 - 0.05 ng/g which is lower than or comparable to VEN extraction from fish tissues reported in other methods in the literature. The validated extraction method was applied to an in-lab exposure of male rainbow darters (Etheostoma caeruleum) collected from a clean reference site in the Grand River, ON. The goal of this exposure was to determine if there was an enantioselective effect on the bioaccumulation of R-VEN and S-VEN in a sentinel small-bodied fish species exposed to a single enantiomer. The fish were exposed to 1 µg/L of R-VEN or S-VEN for up to 14 days, with samples being collected on days 0, 1, 4, and 14. S-VEN appeared to bioaccumulate in fish more than R-VEN, as S-VEN was significantly higher in fish tissue after 4 and 14 days of exposure. The metabolite S-desVEN was also found in fish exposed to S-VEN for 4 and 14 days, which suggests the metabolism of S-VEN into S-desVEN in fish, and subsequent accumulation and/or retention (there was no R- or S-desVEN detected in the water). In contrast, R-desVEN was not found in fish exposed to R-VEN at any time point. Subtle differences in bioaccumulation of VEN and desVEN enantiomers in fish were observed but further studies are needed to determine if there is an enantiomeric shift during bioaccumulation that would alter risk in wild fish.
Microbial ecology of nitrification in engineered water treatment systems
(University of Waterloo, 2025-01-20) McKnight, Michelle Marie; Neufeld, Josh
Nitrification is performed primarily by chemolithoautotrophic microorganisms and is important for nitrogen transformation in aquatic environments. The understanding of nitrification has evolved over the years from a previously understood two-step process mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) that perform ammonia oxidation to nitrite, and nitrite-oxidizing bacteria (NOB) that perform nitrite oxidation to nitrate. More recent research has revealed the existence of complete ammonia-oxidizing (“comammox” or CMX) bacteria from the genus Nitrospira that are capable of oxidizing ammonia to nitrate. With three groups of ammonia oxidizers often existing in the same environment, research is needed to understand the microbial ecology of nitrifying communities. These ammonia oxidizers play important roles in engineered systems, including wastewater treatment plants (WWTP) and aquarium biofilters, where they transform ammonia waste (NH₃/NH₄⁺) to less toxic nitrate (NO₃⁻) via nitrite (NO₂⁻). Prior to the discovery of comammox Nitrospira, previous research revealed that AOA dominated over AOB in freshwater aquarium biofilters. In Chapter 2, aquarium biofilter microbial communities were profiled and the abundance of all three known ammonia oxidizers were quantified using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquariums. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters, except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results indicate that comammox Nitrospira play a previously overlooked but important role in home aquarium biofilter nitrification. Following the identification of comammox Nitrospira among dominant ammonia oxidizers in freshwater aquarium biofilters, Chapter 3 monitored microbial community succession and water chemistry for three independent home aquariums during their first 12-weeks after start-up, with weekly collection of biofilter beads and sponge samples. Extracted DNA from biofilter samples was used for 16S rRNA gene sequencing to determine microbial community composition and quantitative PCR (qPCR) to quantify ammonia monooxygenase (amoA) genes. Water samples were also collected weekly for measurements of ammonia, nitrite, and nitrate. Biofilter nitrification activity reduced ammonia and nitrite concentrations below detectable limits by week 3 in two of the three aquariums, which showed comparable nitrification activity by the week 8 time point. Detection of ammonia oxidizers by qPCR coincided with ammonia oxidation activity for all systems. The two aquariums with nitrification occurring by week 3 contained live plants, whereas the aquarium with delayed nitrification did not, suggesting that live plants might provide an effective nitrifier inoculation source for aquarium establishment. Additionally, a preference in biofilter material was observed for detected AOA, which were present in higher abundance in bead samples compared to sponge samples. Although the tested aquaria differed in the timing and prevalence of ammonia oxidizers in biofilters during community establishment, samples from all three aquaria were consistently dominated by comammox Nitrospira by the end of 12 weeks. Additional metagenomic functional profiling of week 12 biofilter samples confirmed the presence of AOA amoA genes and comammox Nitrospira amoA genes as detected by qPCR for all aquaria, with nitrite oxidation marker gene nxrB for both comammox Nitrospira spp. and canonical NOB Nitrospira spp. also detected. Although this work sheds light on how ammonia oxidizers establish in residential freshwater aquaria, further research is needed to test factors that impact the establishment of nitrifiers populations, such as inoculation sources, fish loads, and water chemistry. Novel WWTP technologies, including membrane aerated biofilm reactors (MABR), aim to improve nitrogen removal, reduce energy consumption, and improve nitrification in cold weather conditions. The municipal WWTP in Southern Ontario was upgraded with an MABR system in 2022, which was installed upstream of the existing conventional activated sludge (CAS) system at a municipal scale. This current study evaluates the impact of an MABR upgrade on the CAS and MABR microbial communities, with this MABR system being the largest in North America based on media surface area at the time of its installation. In Chapter 4, the microbial community of the CAS system was characterized before and after the MABR upgrade to evaluate the impact of the upstream MABR installation on the functional potential of the downstream activated sludge. Microbial community characterization was done using 16S rRNA gene amplicon sequencing of the V4-V5 region and additional MABR biofilm samples were characterized using metagenomic analysis to evaluate the functional potential of the biofilm for nitrification and denitrification. Before upgrade, the CAS system included the nitrifiers Nitrosomonas (AOB) and Nitrobacter (NOB), which exhibited seasonal abundance and activity differences. Following the upgrade to the hybrid MABR-CAS system, seeding effects from the MABR biofilm increased diversity of the CAS, including nitrifiers. Along with AOB, Nitrospira NOB and comammox Nitrospira were present in the MABR. Metagenomic profiling showed that biofilm microbial communities were well equipped to perform nitrification, denitrification, and phosphate removal. Characterization of the microbial communities in the plant showed that the MABR technology had a positive impact in increasing microbial diversity in this treatment system, along with an increased inventory of nitrifiers with diverse metabolic capabilities.
Discovery and characterization of novel biofilm-associated proteins in Pseudoalteromonas tunicata
(University of Waterloo, 2025-01-17) Ali, Sura; Doxey, Andrew
Pseudoalteromonas tunicata is a marine bacterium that is a useful model for studying mechanisms of biofilm development due to its ability to form, colonize, and inhibit growth of other microorganisms in marine and eukaryotic host-associated biofilms. However, the pathways responsible for P. tunicata biofilm formation are still incompletely understood, in part due to a lack of functional information for a large proportion of its proteome. In this thesis, I use comparative shotgun proteomics to explore P. tunicata biofilm development from the planktonic phase to three stages (early, middle, late) of biofilm development. Proteomic analysis identified 232 proteins that were up regulated during different stages of biofilm development, including proteins known to be important for P. tunicata biofilm development (e.g., autocidal enzyme AlpP, violacein proteins, and various pili proteins) as well as many hypothetical proteins of unknown function. I then characterized two novel, biofilm-associated hypothetical proteins, labeled EAR28894 and EAR30327. Functional characterization of EAR28894 revealed that it is the major S-layer protein of P. tunicata. Bioinformatic methods predicted a beta-helical structure for EAR28894 similar to the Caulobacter S-layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S-layer was found surrounding the outer membrane in wild-type cells and completely removed from cells in an EAR28894 deletion mutant. S-layer material also appeared to be “shed” from wild-type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S-layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio and found exclusively in marine metagenomes. This novel protein family was given the name Slr4. Functional investigation of the uncharacterized protein, EAR30327, revealed its function as a novel biofilm adhesin. This protein, which I designated as BapP, was the top identified biofilm-associated protein by proteomic analysis. BapP showed partial homology to outer membrane adhesins containing repeats of bacterial cadherin-like and immunoglobulin (Ig) domains. A ΔbapP mutant strain was unable to form proper pellicle biofilms in liquid media. The Δ bapP mutant also had a significantly reduced ability to form biofilms in crystal violet assays, which was rescued by re-insertion of the bapP gene into the genome. As predicted by the identification of putative Ca2+-binding motifs in BapP, biofilm formation in the wild-type strain was demonstrated to be Ca2+-dependent, which was significantly reduced in the ΔbapP mutant. This study provides a unique proteomic dataset of biofilm development and identifies BapP as a Ca2+-dependent adhesin responsible for biofilm formation in P. tunicata. The occurrence of BapP-related homologs in other species suggests that this protein family represents a broadly conserved mechanism for biofilm adhesion in marine Gammaproteobacteria species. This thesis research establishes a proteomics-based pipeline for biofilm protein discovery and new directions for biofilm research in P. tunicata and related bacteria, and offers insights into potential targets for biofilm management and control.
Comparing different delivery systems in site-specific genome editing in Chlorella vulgaris
(University of Waterloo, 2025-01-14) Ahmadi, Parisa; Ward, Valerie
Microalgae, such as Chlorella vulgaris, are among the most important eukaryotic microorganisms due to their ecological, biotechnological, and sustainable energy applications. Although the genome of C. vulgaris has been sequenced, its full genetic potential remains underutilized due to challenges in efficient genome editing. In this research, we aim to optimize the CRISPR-Cas9 genome editing system to achieve site-specific knockouts in the C. vulgaris genome. Two different delivery methods for CRISPR components plasmid-mediated transformation and direct ribonucleoprotein delivery were compared in terms of efficiency. Three guide RNAs targeting the nitrate reductase gene were designed and tested. The objectives were to assess mutation efficiency, validate phenotypic changes, and develop a genetic toolbox for C. vulgaris, enhancing its potential for applications in biotechnology. Phenotypic analysis confirmed successful gene disruption in transformants, while sequencing results highlighted the challenges in genotypic confirmation, emphasizing the need for improved protocols. This study contributes to advancing genetic engineering tools for C. vulgaris, providing a foundation for future biotechnological applications.
Measurements of water isotope composition and depth variation reveal strong influence of hydroclimate on water balance and water levels of shallow lakes in the Peace-Athabasca Delta (northern Alberta, Canada)
(University of Waterloo, 2024-12-18) Imran, Arisha; Hall, Roland; Wolfe, Brent
Shallow water bodies are abundant, productive ecosystems in northern high-latitude regions where they provide habitat for fauna and hold socio-economic and cultural importance for remote communities. In northeastern Alberta, the Peace-Athabasca Delta (PAD), located within Wood Buffalo National Park (WBNP), is a large (~6000 km2) inland freshwater-rich landscape with an abundance of shallow lakes that is listed as a Ramsar Wetland of International Importance and contributes to WBNP’s UNESCO World Heritage status. However, concerns have long persisted about the effects of primarily hydroelectric regulation of Peace River flow and climate change on widespread drawdown of lake water-levels in the delta. In response to these concerns, and pressures from local communities and an international agency, a federal Action Plan was developed with goals to improve aquatic ecosystem monitoring at the PAD. Despite considerable prior study, this requires better understanding of hydrological processes that influence lake water balances and water levels in the delta and advancement of methodological approaches. A 7-year research project was launched in 2015 to inform the design of a long-term aquatic ecosystem monitoring program at the PAD. Research presented in this thesis is an outcome of two key observations that were made during the last two years (2020-2021) of that project, which provided opportunity to explore the influence of unusually high relative humidity on isotope-based lake water-balance modelling and the influence of snowmelt runoff on lake depth when widespread ice-jam flooding occurred. For two decades, the isotope composition of an index lake (informally named ‘PAD 18’) provided a constant and reliable estimate of δSSL (i.e., the water isotope composition of a terminal basin where evaporation = inflow), a key parameter used in an isotope framework for water-balance modelling of lakes at the PAD. In 2020-2021, a sudden and unprecedented shift occurred in the isotope composition of PAD 18, which required closer examination of the isotope framework to ensure accurate assessments of lake water balances. The shift in isotope composition of PAD 18 coincided with an increase in relative humidity by 4.6% from 66.2% (2015-2019) to 70.8% (2020-2021). An independent calculation of δSSL, using formulation by Gonfiantini (1986) and flux-weighted air temperature and relative humidity from 2020-2021, was determined to be equivalent to the observed isotope composition of PAD 18 during 2020-2021, confirming the shift in isotope composition reflected re-equilibration of the lake water with the atmosphere following the rise in relative humidity. The importance of accurate characterization of δSSL for isotope mass-balance modelling is demonstrated by estimating evaporation-to-inflow (E/I) ratios for 61 lakes across the PAD in July 2021 using the previous 2015-2019 isotope framework and the new 2020-2021 isotope framework. Results show differences between the two frameworks are minor when E/I ratios are low, but they become consequential when E/I ratios are high because of the non-linear effect of relative humidity on isotope framework parameters. Appropriate parametrization for isotope mass-balance modelling is critical for monitoring agencies utilizing isotope-based techniques at the PAD, and elsewhere. Thus, re-evaluation of isotope framework parameters may become increasingly important as climate warming fosters greater variability of meteorological conditions. Snowmelt runoff is well-recognized for sustaining shallow waterbodies across semi-arid and sub-humid northern regions where evaporation rates are high. At the PAD, however, the influence of snowmelt runoff from within the delta remains largely unknown because the attention on drawdown of perched lakes has focused almost exclusively on alteration of the ice-jam flood regime. During spring of 2020, extensive ice-jam flooding occurred which raised water levels in flooded lakes across the PAD, but water levels rose also in many non-flooded lakes, providing an opportunity to explore the role of snowmelt runoff on lake levels. Using measurements of water depth and water isotope compositions, the contributions from snowmelt runoff and river floodwaters to lake-level rise were quantified at 52 lakes and spatial patterns were explored. Results reveal that the lake-level rise from snowmelt runoff at non-flooded lakes rivals the rise caused by floodwaters at flooded lakes. Geospatial analyses show lake-level rise by snowmelt was greatest in the northern Peace sector and the southwestern margin in the southern Athabasca sector where greater topographic relief and denser vegetation enhance snow accumulation via redistribution of snow by wind from adjacent flat, sparsely vegetated terrain. In contrast, the rise due to floodwater input was greatest in lower lying flood-prone central areas of the Athabasca sector. Exploration of meteorological records since 1963 reveal that similar contributions of snowmelt runoff to lake-level rise as occurred in 2020 were likely common between the early 1960s and late 1980s but only once since 1987 (2018), suggesting that reduced input from snowmelt runoff has contributed to drawdown of shallow perched lakes in recent decades. Concern for drawdown of water levels in the PAD have prompted a need for enhanced monitoring of lake water balances and water levels, especially as mitigative actions are considered. The research presented in this thesis advances knowledge about the influence of shifts in relative humidity on isotope-based modelling of lake water balances and the role of snowmelt runoff on lake depth. The novel integration of water isotope composition and water depth measurements demonstrated here provides opportunity for assessing the success of mitigative actions such as strategic water releases from the W.A.C. Bennett Dam located upstream of the delta to enhance ice-jam flooding. While long-term aquatic ecosystem monitoring programs must be built on a foundation of knowledge generated through primary research, systematic and repeated measurements over sufficient scales of space and time may also trigger new research that leads to important new discoveries, as revealed in this thesis.
Glyphosate as a Source of Phosphorus for Bacteria: Selection of an Enrichment Culture for Glyphosate Degradation, and Analysis of the C-P Lyase Pathway
(University of Waterloo, 2024-12-17) Van Dyk, Adrian; Charles, Trevor
Glyphosate is one of the world’s most widely used herbicides, the active ingredient in Roundup as well as many generic herbicide brands. Though it is one of the safer herbicides, its ubiquity necessitates further study of the pathways that bacteria can use to degrade it. This study investigates the ability of bacteria to break the strong carbon-phosphorus bond of glyphosate. The carbon-phosphorus bond has the highest activation energy of the bonds within glyphosate, and breaking this bond is therefore a rate-limiting step of complete glyphosate mineralization. By optimizing a defined media and supplementing with glyphosate as the only source of phosphorus, the ability of bacterial species to grow on glyphosate as the sole source of phosphorus can be tested and compared to their growth when easily accessible ortho-phosphate is provided. This defined media was used to create an enrichment culture to study which species are selected for by glyphosate as the sole source of phosphorus, and compare them to cultures selected on easily accessible ortho-phosphate. Glyphosate as sole source of phosphorus selected for a different suite of species, especially those in the Rhizobiaceae family, than ortho-phosphate selected for. The Rhizobiaceae genus Agrobacterium was found to contain particularly good degraders of glyphosate, and a deletion and replacement experiment revealed that the C-P lyase pathway is required for glyphosate degradation in an Agrobacterium pusense isolate. Genome and growth comparisons between A. pusense, A. tumefaciens, and A. fabrum isolates revealed them all to be able to degrade glyphosate, and that they share very similar C-P lyase operons, despite having significant heterogeneity in their genomes. Additionally, a commercial enzyme linked immunosorbent assay was used to quantify glyphosate degradation. This work adds support to the theory that C-P lyase is required for glyphosate degradation in Agrobacterium, and sets the stage for additional research into glyphosate degradation pathways by bacteria.
Trait selection and the adaptive potential of 𝘗𝘪𝘤𝘦𝘢 𝘮𝘢𝘳𝘪𝘢𝘯𝘢 in the face of climate change
(University of Waterloo, 2024-12-04) Henry, Sabina; Messier, Julie
The local persistence of long-lived organisms is at risk as climate change drives a rapid shift in selection regimes world-wide. Although adaptive evolution is one of the main mechanisms by which populations persist in changing environments, we have little information regarding how selection regimes will shift in response to continued climate change, nor on the potential for trees to evolve adaptively under novel selection pressures. To address these gaps, here we assessed the changes in selection in three sites along a spatial climate gradient which mimics expected temporal changes in climate and determined whether trait covariance might accelerate or impede the rate of adaptive evolution of seven P. mariana populations in the warmer and drier environment. In three common garden sites established 50 years ago, we measured an array of traits which represent water use, response to temperature, structural investment, and metabolic efficiency. Our findings reveal that all 10 traits measured in this study were under selection in at least one site. We also find different traits are under selection in each site, with four instances where the shift in selection gradient is consistent with shifts in climate: water use efficiency (WUE); needle carbon to nitrogen ratio (CN); the interaction between WUE and CN; and the interaction between CN and huber value. In the warm and dry site, traits under selection were largely uncorrelated, with only four of the 49 trait combinations under selection exhibiting intra population trait covariances. The shifts in selection gradient suggest that climate change may select for needles with higher WUE, higher structural carbon and higher hydraulic supply to the needles. The few trait-trait correlations indicate that phenotypic integration should neither impede nor facilitate adaptive evolution, leaving P. mariana provenances with the evolutionary flexibility to respond to climate change regardless of the direction to selection.
Time trends in abundance and composition of microplastic particles deposited in profundal sediment of two headwater reservoirs within the Grand River Watershed (Ontario, Canada)
(University of Waterloo, 2024-11-20) Watson, Meredith; Hall, Roland
Plastic pollution has become pervasive in the environment, raising concern for degradation of aquatic ecosystems by microplastics (MPs). Studies on the supply and abundance of MPs in Canadian freshwaters are rapidly emerging, however temporal trends spanning several decades remain sparse. In this study, we report multidecadal records of MP abundance and composition in dated sediment cores from two headwater reservoirs (Belwood Lake and Conestogo Lake) located within an agricultural region of the rapidly urbanizing Grand River Watershed (GRW; southern Ontario, Canada), a major tributary of Lake Erie. Extracted MPs from contiguous 1-cm thick intervals of sediment cores were enumerated and categorized by shape (fragment, fiber). A subset of samples at approximately decadal intervals were chemically characterized using Laser Direct Infrared (LDIR) Spectroscopy. Results reveal that MP concentrations in both reservoirs varied within a similar range (~50-550 particles g/dw) with no observed increasing trend since the start of each record (1957 for Belwood Lake, 1985 for Conestogo Lake). MP flux in Conestogo Lake increased from ~50 particles/cm^2/year in the mid-1980s to ~100 particles/cm^2/year in recently deposited sediment, whereas MP flux varied without a trend between ~5-60 particles/cm^2/year in Belwood Lake since 1957, apart from a peak in the uppermost sample (~150 particles/cm^2/year). More rapid sediment deposition at Conestogo Lake accounts for the difference in MP flux between the reservoirs, suggesting reduction of sediment transport could reduce the supply of MPs to aquatic ecosystems in the GRW. Analysis by LDIR revealed that polyamide, rubber, and polyethylene were the most abundant polymer types in both reservoirs. The relative abundance of rubber particles has increased since the 1990s, indicating an increase of paved road surface and vehicle traffic as a potential source. The findings suggest MP accumulation in these upstream rural reservoirs of the GRW may be primarily driven by local, regional, and hydrologic factors instead of the rise in global plastic production.
RNA-seq exploration of host responses and viral genomic diversity in SARS-CoV-2 infection
(University of Waterloo, 2024-11-18) Luc, Jessica; Doxey, Andrew
Since the discovery of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there have been over 700 million cases and over 7 million deaths worldwide. Although some patients develop severe symptoms such as multiple system organ failure, others can be asymptomatic. This reflects the role of the immune system in disease progression. Although there exist standard PCR and antigen-based tests for SARS-CoV-2 detection and diagnostics, an important research goal is the identification of predictive biomarkers that are indicative of COVID-19 severity. In this thesis, I performed bioinformatic analyses of two original RNA-seq datasets generated by collaborators to gain insights into the mechanisms and host responses of SARS-CoV-2 infection. The first dataset consisted of transcriptomic data from SARS-CoV-2 infected human lung cells as well as bat (Eptesicus fuscus) derived cells, providing insights into host responses of multiple mammalian species. The second dataset consisted of transcriptomic data from nasopharyngeal swabs collected from COVID-19 patients. A bioinformatic approach was used in both datasets to identify host genes that were up-regulated and down-regulated by SARS-CoV-2 infection as well as to identify biologically relevant viral mutations. The analysis resulted in the discovery of a novel R685P mutation in the SARS-CoV-2 spike glycoprotein, that had increased frequency in the Eptesicus fuscus derived cell line. Variant analysis from the clinical nasopharyngeal swab RNA-seq dataset also revealed the existence of a SARS-CoV-2 quasispecies containing multiple distinct viral genotypes with a unique population structure in each patient. Despite each dataset being distinct, a similar bioinformatic analysis was applied to both datasets to reveal host response patterns and visualize the evolution and mutational spectrum of SARS-CoV-2. Although COVID-19 is no longer considered a global emergency, furthering our knowledge of the SARS-CoV-2 infection can aid in the development in new therapeutics and diagnostic tools.
Metal Concentrations in an Arctic Wastewater Wetland: Insights and Innovations from Baker Lake, Nunavut, Canada
(University of Waterloo, 2024-11-14) McPhedran, Bronte; Swanson, Heidi; Hanson, Mark
The unique climatic conditions and isolated geographies of Arctic communities present distinct challenges for municipal wastewater treatment. Most communities depend on natural wetlands to provide passive treatment of wastewater; however, increased pressure on these systems from prolonged use, climate change, and intensifying human activity has resulted in the recent development of northern-specific standards for wastewater. These standards, among other objectives, aim to maintain healthy fish populations and diverse aquatic ecosystems, while calling for community consultation, integration of traditional knowledge, and adherence to federal and territorial regulations. A passive wastewater treatment system near Baker Lake, NU, served as a case study to address knowledge gaps regarding ecosystem function and trace metal dynamics in abiotic and biotic ecosystem compartments subject to wastewater inputs. Additionally, this research explored a method for time-resolved analysis of contaminant histories, which is particularly valuable in environments lacking baseline data or where regular monitoring is challenging, including in the Arctic. Between the summers of 2019 and 2021, a series of lakes that represented wastewater-affected and reference systems were sampled for water, sediment, and fish. Water from wastewater-affected lakes had elevated concentrations of metals (e.g., Mn, Fe, Cu) and increased primary productivity (as inferred from chlorophyll-a concentrations), with persistent hypereutrophic conditions; extremely low dissolved oxygen levels were recorded in the lake closest to wastewater influent. Although sediment analysis near wastewater sources indicated higher levels of Cu and Zn, overall sediment chemistry was similar across all sites. The influence of wastewater on the trophic ecology of Burbot (Lota lota) was minimal, whereas Arctic grayling (Thymallus arcticus) had more enriched (i.e., less negative) δ13C at wastewater-affected sites, suggesting altered energy pathways in lakes that receive nutrients from wastewater. Analyses of metals in muscle tissue revealed lower, although not significant, concentrations of methylmercury – the toxic, biomagnifying form of mercury - in fish from wastewater-affected sites compared to those from reference sites. These lower concentrations of methylmercury in fish muscle tissue occurred despite higher concentrations in abiotic media, potentially because of somatic growth dilution, but more research is necessary. Analyses of trace metals in otoliths revealed distinct multi-element otolith signatures (i.e., Zn, Mn, Cu, Fe, and Pb) between wastewater-affected and reference lakes, and significantly greater concentrations of Fe in a lake receiving wastewater across two reconstructed periods of growth. Together, findings from this research not only fill knowledge gaps regarding the influence of municipal wastewater on cold, oligotrophic freshwater systems in the subarctic, but also highlight the potential uses and limitations of otoliths as markers of wastewater exposures.
Microbiology of bentonite clay relevant to a deep geological repository for used nuclear fuel
(University of Waterloo, 2024-11-12) Beaver, Rachel; Neufeld, Josh
Nuclear energy is an important source of energy globally, but results in the production of used nuclear fuel. When removed from a nuclear reactor after its useful lifetime, used nuclear fuel is still highly radioactive and must be stored safely for approximately one million years until it returns to the radioactivity level of naturally occurring uranium ore. Canada, along with other countries that have an inventory of used nuclear fuel, is in the process of designing a deep geological repository (DGR) for long-term storage of used nuclear fuel. In this system, used nuclear fuel, itself a stable solid, will be stored in copper-coated steel used fuel containers, and placed in bentonite clay buffer boxes made of highly compacted Wyoming MX-80 bentonite. Buffer boxes would then be stacked in placement rooms approximately 500 m below ground in a suitable host rock with spaces between buffer boxes and host rock filled in with a granulated form of bentonite referred to as gapfill material. To ensure the longevity of a DGR, it is important to consider the role that microorganisms could play, particularly through contributing to corrosion of used fuel containers through a process termed microbiologically influenced corrosion. Expected to dominate occurrences of microbiologically influenced corrosion under anoxic conditions, sulfate-reducing bacteria (SRB) are a primary target of research, but rarely live in isolation, thus necessitating the study of microbial communities that could live in the bentonite clay surrounding used fuel containers on a broader scale. Due to the potentially detrimental role that some microorganisms could play in a DGR, a goal in designing a DGR is to compact bentonite, a type of swelling clay, to a sufficiently high dry density that microbial growth is suppressed upon saturation. The first goal of this thesis was to investigate the bentonite dry density necessary to suppress microbial growth in Wyoming MX-80 bentonite under oxic conditions and gapfill material under both oxic and anoxic conditions. A set of pressure vessel experiments demonstrated suppression of microbial growth under oxic conditions in bentonite compacted to a minimum dry density of 1.4 g/cm3. Under anoxic conditions, growth of heterotrophs was suppressed in pressure vessels with a minimum dry density of 1.45 g/cm3, but culturable SRB persisted in abundances higher than the as-received bentonite starting material in the outer layers of all pressure vessels throughout the full one-year experiment. Additional experiments were conducted to explain the increase in abundance of SRB, which had not previously been observed in other studies. These experiments suggested that the increase in abundance of SRB was likely not inherent to the gapfill material, nor was there evidence for it being a result of differences in SRB medium or in amounts of trace oxygen between studies. In both oxic and anoxic pressure vessels, an initial increase in abundance of culturable heterotrophs was observed prior to complete saturation, presumably as water became available but swelling pressure remained sufficiently low to allow for their growth. Although previously proposed to potentially be associated with a recovery from the viable but not culturable state rather than growth, a follow-up experiment suggested that the initial increase in abundance of culturable heterotrophs was likely a reflection of growth (i.e., cell division). Dry density is an important consideration in DGR design, but it is not the only physical property that could influence microbial growth within a DGR. Temperature is expected to fluctuate from natural subsurface temperatures of <20°C to temperatures as high as 94°C, and little research has been conducted to explore the potential for microbial growth in bentonite at these elevated temperatures. This thesis includes experiments testing the abundance and community composition of microorganisms adapted to a variety of temperatures in as-received and hydrated bentonite. The results showed a low abundance of culturable microorganisms that survived incubation at 60°C, but 16S rRNA gene profiles dominated by presumably unculturable representatives of the thermophilic family Thermoactinomycetaceae. Hydrated bentonite was additionally incubated at temperatures of 75, 90, and 105°C, but DNA sequencing results did not show a shift in community composition from as-received bentonite, suggesting that the natural as-received bentonite microbial community may not include members adapted to these very high temperatures. Lab-scale experiments allow for testing of very specific DGR-relevant conditions (e.g., dry density and temperature) and the effect these have on microbial community abundance and composition. However, a DGR is being designed to exist for upwards of one hundred thousand years, which is not a realistic timescale for any experiment. To circumvent this limitation, one approach is to couple lab-scale experiments to the study of natural analogues, which have naturally existed for DGR-relevant timeframes. This thesis presents a study of the Tsukinuno bentonite deposit in Japan, which can serve as a natural analogue to DGR bentonite. In this study, sequencing of DNA extracted from bentonite revealed microbial communities dominated by sequences associated with Thiobacillus, Hydrogenophaga, Comamonadaceae, and Pseudomonas. Although differences in community composition were observed between samples, microbial communities were relatively similar for all four studied cores and at all depths into the clay bed. A series of geochemical parameters were measured to help identify factors that may influence microbial community composition. The abundance of culturable anaerobic heterotrophs was positively correlated with the concentration of nitrate, which could be used by anaerobes for denitrification, and the abundance of culturable aerobic and anaerobic heterotrophs was negatively correlated with the abundance of the clay mineral montmorillonite, increased concentrations of which would increase the swelling capacity of the bentonite. The results presented throughout this thesis will together be useful for incorporation into future models of microbial activity within a DGR and can ultimately be used to inform DGR design.
The evolutionary and ecological factors that shape ectoparasite populations and communities at multiple scales
(University of Waterloo, 2024-11-12) Sauk, Alexandra; Broders, Hugh
Although parasites are one of the most prolific and diverse consumer groups on the planet, they are often excluded from biodiversity surveys as they are difficult to detect and identify. This deficit limits our understanding of host-parasite relationships and parasite diversity. The vast diversity of host-parasite relationships means that many ecological and evolutionary forces may be at play, shaping the evolution of host and parasite in different ways and varying between species pairs. Bats and their ectoparasites provide a unique system to study the factors influencing parasite populations and communities. The different ecological niches and social behaviours of bats provide variation in the ectoparasites they encounter, and the selective forces experienced by the ectoparasites. I hypothesised that certain attributes of ectoparasite infections (e.g., ectoparasite diversity and infection level) are influenced by the life history traits of both host and ectoparasite and be the environmental restrictions of individual ectoparasite species. Using a collection of ectoparasites passively collected from bats throughout Atlantic Canada between 1999 and 2017, I quantified the ectoparasite communities of two bat species, Myotis lucifugus and M. septentrionalis, and used model-based inferences to assess the differences in infection of their two most common ectoparasites, the mite Spinturnix americanus and the flea Myodopsylla insignis. I found that both bat species had similar ectoparasite communities while S. americanus and M. insignis showed opposing trends in presence and abundance between the two bat species, in keeping with their different life history strategies. I also used a subset of this collection to compare how life history traits and host-parasite dynamics influence the genetic structure and biogeography of co-infecting ectoparasites. I found limited genetic structure with M. insignis exhibiting some isolation by distance between Labrador and Nova Scotia and S. americanus exhibiting regional differentiation between the island of Newfoundland and the mainland. I also provide a synthesis of the currently described bat ectoparasites in North America and an analysis of how host characteristics and environmental factors influence ectoparasite richness and geographic distribution. I found that estimated ectoparasite richness varies widely between host species but is influenced by sampling effort. Bat ectoparasite diversity appears consistent with the predictions of the latitudinal diversity hypothesis with a 3.4% decrease in species richness for every degree increase in latitude. Overall, my findings add to the evidence that ectoparasite populations and communities are shaped by life history traits of the host and ectoparasite. I suggest multidisciplinary collaborations between bat biologists, parasitologists, and taxonomists are necessary to collect ectoparasites and catalogue bat-ectoparasite associations to better understand the ecological and evolutionary forces that shape these communities and to better be able to conserve them in the face of ongoing threats from climate change and landscape changes.
Predicting Black Carp (Mylopharyngodon piceus) age at sexual maturity with water temperature
(University of Waterloo, 2024-09-24) Wu, Jiayi; Cuddington, Kim
Black Carp is one of the four invasive Asian carps in North America. It is currently considered established in the Mississippi River and has the potential to invade into the Great Lakes watershed. Black Carp is known for its ability to consume freshwater unionids, and thus could bring potential changes to the benthic ecosystems. For effective management and mitigation strategies, it is critical to understand its likely population growth rate in new environments, and to be able to predict the environmental conditions of the potential habitats it may become established in. Age at sexual maturity of the population has been identified as an important variable in determining the population establishment speed, as faster maturation typically corresponds to faster population growth. I demonstrated that temperature metrics such as air temperature can be used to predict the age of maturity of Black Carp. Using age at maturity data and related climate metrics around the world, I found that winter duration and winter air temperature are the two best predictors of Black Carp age at maturity. This suggests that winter can play an important role in the maturation process of this invasive species. Populations experiencing longer and colder winters tend to mature later. While temperature may be an important predictor for the speed of Black Carp invasion, water temperature data is not always available for regions of interest. Using empirical water temperature from 12 tributary locations in the Great Lakes watershed, I established a simple statistical model to predict water temperature during the growing season. I found that simple regression-based models using air temperature as input can perform better than a complex processed-based global model for estimating water temperature in the Great Lakes region.
Effect of Riparian Forest Cover on Cellulose Decomposition in Agricultural Streams
(University of Waterloo, 2024-09-24) Hewitt, Kristen; Yates, Adam
Agricultural practices often result in human impacts to aquatic ecosystems. The presence of riparian forests has been shown to mitigate some of these impacts, however, there is limited knowledge of how riparian forests can mitigate human impacts on microbial community function. This study investigates effects of riparian forest cover on in-stream cellulose decomposition. The study used the cotton strip assay to compare two sets of streams in summer, fall, and winter seasons to assess the association between riparian forest cover and cellulose decomposition in summer in agricultural catchments. Results from summer and autumn show there is greater cellulose decomposition in streams with riparian forest cover than streams without riparian forest cover. Cellulose decomposition also increased linearly with riparian forest in the summer. Decomposition in the assessed streams was also compared to cellulose decomposition in reference streams within the same region. Significant differences were observed between reference and non-forested streams, but not reference and forested streams, in the summer. These differences were not explained by typical environmental parameters (e.g., temperature, nutrients). These results suggest that riparian forest cover influences the decomposition by protecting organic matter processing in streams, though the specific threats against which riparian forests provide protection remain unclear. In future studies, considering agricultural contaminants (e.g., pesticides, pharmaceuticals) and investigating the differences in microbial community composition between groups may help close this knowledge gap.
Ploidy impacts the response of Chinook salmon (𝘖𝘯𝘤𝘰𝘳𝘩𝘺𝘯𝘤𝘩𝘶𝘴 𝘵𝘴𝘩𝘢𝘸𝘺𝘵𝘴𝘤𝘩𝘢) to pathogen and thermal stress
(University of Waterloo, 2024-09-23) Cadonic, Ivan; Craig, Paul
As climate change continues to reduce wild salmon populations, global reliance on aquaculture will become increasingly essential to sustain food availability. However, the environmental implications of aquaculture in causing Pacific salmon stock declines are becoming more apparent and as such the industry needs to transition to practices that would reduce negative impacts on wild fishes. Widespread utilization of sterile triploid fishes offers an alternative to normal diploid fish since they are unable to breed with wild population and are less likely to interact with wild fishes. Triploid salmonids have an extra set of chromosomes that prevent them from producing viable gametes, while also maintaining flesh quality for longer due to lack of nutrient deposition from muscle to eggs during sexual maturation. When farmed under optimal conditions, triploid fish perform similarly to diploids; however, when conditions become suboptimal (e.g. high temperature & infection with pathogens) triploids have reduced survival. The reasons for this reduced performance are mostly unknown, so this thesis aimed to investigate the molecular impacts of increased ploidy in Chinook salmon (Oncorhynchus tshawytscha). Specifically, the response of microRNA (miRNA) and protein coding mRNAs were investigated before and after exposure to pathogenic and thermal stress. MiRNAs are small, non-coding RNAs that bind to specific mRNA molecules and reduce protein translation. I hypothesized that diploid and triploid salmonids have subtle differences in molecular (miRNA, mRNA) indices that are exacerbated by changes in the environment (pathogens and temperature), which results in their poorer performance in response to stress. Chapter 2 investigated the miRNA responses in three immune tissues (hindgut, head kidney, and spleen) before and after exposure to Vibrio anguillarum, a common pathogen Chinook experience in open-sea net pens. Overall, ploidy had minimal impacts on miRNA abundances prior to stress. Abundance of miRNAs were altered in both ploidies after Vibrio exposure to support immune function. Just prior to Vibrio-induced mortality, the spleen of triploid fishes had altered miRNA abundances which identified that in triploids, the spleen is likely associated with their reduced immune competence. Chapter 3 investigated the immune mRNA responses in gill and heart ventricle before and after recovery from acute thermal stress. Triploid gills likely have altered pathogen sensing based on reduced pattern recognition receptor mRNA abundance. Tissue specific alterations after thermal stress provided insight into immune dynamics during a cellular heat-shock response. Specifically, pro-inflammatory cytokine mRNA abundance was elevated after recovery from thermal stress. The immune genes measured are likely thermally responsive in a time-dependent manner based on their variability after recovery from thermal stress. Chapter 4 investigated the cardiorespiratory impacts of increased ploidy before and after a critical thermal maximum (CTmax) trial. In agreement with previous studies, triploid hearts became arrhythmic at lower temperatures indicating that their hearts are less resilient to acute changes in temperature. Additionally, ventricle expression of cardiorespiratory genes was altered in triploid fish, implicating that this tissue is associated with their reduced thermal performance. Overall, this thesis provides evidence for the molecular dysregulation associated with increased ploidy in Chinook salmon. These impacts are minimal under normal conditions but exacerbated by pathogenic and thermal stress. The results here provide targets for aquaculture to develop breeding programs or therapeutics that can be used to improve health and well-being of salmonids, including triploids, in response to climate change.
Genome-encoded metabolic potential of the Nitrosocosmicus genus and related ammonia-oxidizing archaea
(University of Waterloo, 2024-09-17) Cornell, Calvin; Neufeld, Josh
Since the discovery of ammonia-oxidizing archaea (AOA), genomic and experimental evidence suggests that mixotrophic growth may complement chemolithotrophic ammonia oxidation for some AOA representatives. Although members of the Candidatus Nitrosocosmicus genus have been implicated in the use of alternate sources of energy and carbon, stronger genomic evidence is needed to support testing of hypotheses with robust experimental design. This study involved analysis of 58 genomes spanning all four known AOA groups (i.e., I.1a, I.1b, 1.1a-associated, thermophilic AOA; ThAOA). The analyses focused primarily on transporters and enzymes, linking the former to potential substrates from the transporter classification database (TCDB) and the latter to KEGG, Carbohydrate-Active enZYmes (CAZymes), and pathways from GapSeq. Correlated with genome size, the results demonstrate that genomes of the I.1b group, including those from the Ca. Nitrosocosmicus genus, showed the highest abundances of exclusive (only found within this group) and preferred (primarily associated with this group) proteins for alternative metabolism, followed by ThAOA, I.1a, and I.1a-associated. There was extensive functional heterogeneity among representative genomes, particularly for Ca. Nitrosocosmicus and Nitrososphaera genera. The group I.1b genomes encoded for the potential to use nitrogenous substrates such as urea, alanine, glycine, asparagine, and glutamine. Although Ca. Nitrosocosmicus genomes were more commonly associated with use of TCA cycle intermediates, such as citrate, malate, oxaloacetate, and succinate, this potential was likewise found in ThAOA and I.1a representatives. Several I.1b representative genomes encoded enzymes for metabolism of ureidoacrylates and carbamate, CAZymes associated with glycoside hydrolases, genes implying oxidation of sulfur and manganese, and polyamine metabolism. However, genes for synthesis and degradation of polyamines were common to many AOA genomes analyzed. Overall, the results suggest that I.1b-associated representatives encode for mixotrophic metabolism, and future research should verify associated substrate predictions experimentally. Given broad distributions of AOA within terrestrial and aquatic environments, these findings have implications for biogeochemical cycling of carbon and nitrogen on a global scale.
The Effect of Corn Starch and Kappa-Carrageenan Probiotic Encapsulation on Growth and Immune Response in Chinook Salmon (Oncorhynchus tshawytscha)
(University of Waterloo, 2024-09-16) Latimer, Maureen; Dixon, Brian
As vaccines have limited efficacy in fish, alternative preventative measures are used to combat infectious diseases in aquaculture, including probiotics. To be effective, a probiotic must remain stable during storage, feeding, and transit through the gastrointestinal tract. Thus, preparing probiotic feed requires the use of a coating agent and several different types have been tested, as some have adverse effects. To better understand the effect of corn starch and kappa () -carrageenan as a combined coating agent on growth and immune performance in Chinook salmon, a growth trial was conducted. Three different treatments were used, including regular feed, regular feed with a corn starch and -carrageenan coating, and probiotic feed with the same coating. The probiotic feed included a mixture of BioPower® PA (Pediococcus acidilactici, strain CNCM MA 18/5M; Lallemand Inc., Canada) and Seed 14 (Limosilactobacillus reuteri LRE2; Probiotical SpA, Italy). Sampling timepoints were at 0-, 1-, 3-, 7-, and 28-days post diet-introduction, and at each sampling timepoint, the weight and length of 6 fish per treatment were measured, and the head kidney, spleen, and hindgut were collected. There was an additional timepoint at Day 42 where the weight and length of the remaining fish in each barrel were recorded, but no tissues were collected. To assess immunological effects of the coating agent, qPCR for IL-1, IL-8, and TGF- was performed on spleen, head kidney, and hindgut samples. In the hindgut, additional genes were assessed to determine changes in gut barrier integrity, including E-cadherin, claudin 15, junctional adhesion molecule 1 alpha, mucin 2, tricellulin, villin 1, and zonula occludens-1. No significant differences in weight or length were observed between treatments measured over 42 days. No significant differences were found in the assessed proinflammatory transcript levels in the spleen or head kidney sampled at 0-, 1-, 3-, 7-, and 28-days post diet-introduction. Transient changes in the regulatory cytokine TGF- were seen in these tissues. In the hindgut, upregulation of all three immune transcripts were seen on Day 7 in both coated treatments, but these changes were transient, with no significant differences seen on Day 28. Transient changes were also seen in gut barrier gene expression, with no significant differences seen between treatments on Day 28. These results suggest that corn starch and -carrageenan as a combined coating agent has no short-term negative effects on the growth or immune status of Chinook salmon and that it can be used to deliver probiotics that will improve the health of fish in aquaculture.
Effects of Trastuzumab on Alternative Splicing in HER2+ BT474 Breast Cancer Cells
(University of Waterloo, 2024-09-12) Piticaru, Benjamin; McConkey, Brendan
Alternative splicing (AS) is a fundamental process that enhances transcriptomic diversity and protein isoform complexity in eukaryotic cells. Deregulation of AS plays a critical role in various diseases, particularly cancer, where erroneous splicing patterns contribute to oncogenesis, tumor progression, and therapeutic resistance. Human epidermal growth factor receptor (HER)2, overexpressed in more than 20% of breast cancers, has clinical relevance in AS. The humanized monoclonal antibody (mAb) Trastuzumab has been used for decades to fight HER2+ breast cancer with positive results. However, there are many unknowns surrounding the mechanism of this drug and the effects it has on alternative splicing. Next generation long read sequencing technologies like Oxford Nanopore allow researchers to sequence full length transcripts. The ability to sequence long intron/exon spans and repeating regions enables long read sequencing technology to provide new insights into the AS patterns of genes. Identifying AS events is important for understanding changes in function and pathways affected by differing conditions (e.g., drug treatment, diseased cells). Software tools tailored to long read data like Long-read Isoform Quantification and Analysis (LIQA), Full-Length Alternative Isoform Analysis of RNA (FLAIR), FLAME, and Nanosplicer enhance data analysis abilities to detect AS events. They analyze sequencing data by mapping reads to genomes, identifying splice junctions, and clustering AS events. These tools then compare AS events between conditions (e.g., treated vs. untreated cells) to pinpoint significant AS variations. Accurate transcriptome sequencing is vital for research in drug development and diagnostics. The optimization of sequencing methods is an ongoing endeavour that requires continuous development. A chapter of this thesis compares the impact of omitting polyadenylation (poly(A)) enrichment in Oxford Nanopore Technologies (ONT)’s direct complementary deoxyribonucleic acid (cDNA) sequencing protocol to improve library prep efficiency and sequencing accuracy. Analysis indicated that excluding poly(A) selection does not negatively affect sequencing metrics but enhances read length and enables the sequencing of histone messenger ribonucleic acid (mRNA). Further, significant differences in poly(A) tail lengths between selected and unselected samples were found, suggesting a bias against shorter tails with enrichment. Gene composition and unique gene identification remain consistent across conditions. These findings support the potential advantages of omitting poly(A) enrichment in transcriptome sequencing while advocating for further validation. With optimized methods, the impact of Trastuzumab on AS profiles in BT474 and SKBR3 cell lines was tested. Using ONT long-read sequencing, significant AS events were identified in response to Trastuzumab treatment. The findings reveal differential isoform expression in genes involved in cellular signaling, RNA processing, and stress response pathways. Notably, nuclear paraspeckle assembly transcript 1 (NEAT1) and pre-mRNA processing factor 38B (PRPF38B) exhibited distinct AS patterns across multiple cell lines, suggesting HER2-mediated regulatory mechanisms. This study underscores the utility of ONT sequencing and high throughput data processing as an effective and efficient means for explaining complex AS landscapes affected by therapeutic treatments.
Structural and Functional Characterization of a Modular Immunoglobulin A Protease from Thomasclavelia ramosa
(University of Waterloo, 2024-08-29) Tran, Norman; Holyoak, Todd
Immunoglobulin A proteases (IgAPs) are a diverse group of enzymes secreted from a wide range of mucosal bacteria. These enzymes have convergently evolved to cleave immunoglobulin A (IgA), the main antibody found on the mucosa, as a means of modulating the bacterium’s relationship with their host tissues. Due to the various biological functions and biochemical properties of these enzymes, the study of IgAPs has provided multifaceted insight into aspects of mucosal immunity, enzyme structure and function, and the structural basis for substrate specificity. Only two of three known IgAP enzyme families have been investigated using an in-depth structural and functional approach. This thesis thus aimed to carry out these analyses on the IgAP from Thomasclavelia ramosa as a representative member of this last poorly characterized family. X-ray crystallographic, small-angle X-ray scattering, and gel-based kinetic techniques were used to reveal that, unlike the other two IgAP families, the T. ramosa IgAP has a truly modular protein architecture that can be split into and produced as distinct minimized domains that retain function. The crystal and solution-scattering structures of various domain constructs were also used to generate a working model for how the T. ramosa IgAP recognizes and has high specificity for IgA. This thesis provides the first in-depth biochemical account of this IgAP family and pave the way for advances in clinically relevant IgAP-related research and our understanding of IgAPs as a whole.

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